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West Nile virus (WNV) is similar to other RNA viruses in that it forms genetically complex populations within hosts. The virus is maintained in nature in mosquitoes and birds, with each host type exerting distinct influences on virus populations. We previously observed that prolonged replication in mosquitoes led to increases in WNV genetic diversity and diminished pathogenesis in mice without remarkable changes to the consensus genome sequence. We therefore sought to evaluate the relationships between individual and group phenotypes in WNV and to discover novel viral determinants of pathogenesis in mice and fitness in mosquitoes and birds. Individual plaque size variants were isolated from a genetically complex population, and mutations conferring a small-plaque and mouse-attenuated phenotype were localized to the RNA helicase domain of the NS3 protein by reverse genetics. The mutation, an Asp deletion, did not alter type I interferon production in the host but rendered mutant viruses more susceptible to interferon compared to wild type (WT) WNV. Finally, we used an in vivo fitness assay in Culex quinquefasciatus mosquitoes and chickens to determine whether the mutation in NS3 influenced fitness. The fitness of the NS3 mutant was dramatically lower in chickens and moderately lower in mosquitoes, indicating that RNA helicase is a major fitness determinant of WNV and that the effect on fitness is host specific. Overall, this work highlights the complex relationships that exist between individual and group phenotypes in RNA viruses and identifies RNA helicase as an attenuation and fitness determinant in WNV. [ABSTRACT FROM AUTHOR]